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Review ARTICLE

Topographical organization of the pedunculopontine nucleus

Medical Research Council Anatomical Neuropharmacology Unit, Department of Pharmacology, University of Oxford, Oxford, UK

Neurons in the pedunculopontine nucleus (PPN) exhibit a wide heterogeneity in terms of their neurochemical nature, their discharge properties, and their connectivity. Such characteristics are reflected in their functional properties and the behaviors in which they are involved, ranging from motor to cognitive functions, and the regulation of brain states. A clue to understand this functional versatility arises from the internal organization of the PPN. Thus, two main areas of the PPN have been described, the rostral and the caudal, which display remarkable differences in terms of the distribution of neurons with similar phenotype and the projections that originate from them. Here we review these differences with the premise that in order to understand the function of the PPN it is necessary to understand its intricate connectivity. We support the case that the PPN should not be considered as a homogeneous structure and conclude that the differences between rostral and caudal PPN, along with their intrinsic connectivity, may underlie the basis of its complexity.

Introduction

The pedunculopontine nucleus (PPN) is located in the upper brainstem and has an irregular shape delimited by the borders of its population of cholinergic neurons. The PPN has been conserved in evolution across species and is present in early mammals and amphibians (Marin et al., 1998; Grillner et al., 2008). Defined in humans in 1982 (Olszewski and Baxter, 1982), it is considered a part of the reticular activating system and, as a reticular structure, it has been argued that PPN has no clear boundaries. One of the main characteristics of neurons of the PPN is their long-range axonal projections, reaching numerous targets across the brain, from distant forebrain structures (Woolf and Butcher, 1986; Hallanger and Wainer, 1988a) to the spinal cord (Rye et al., 1988; Spann and Grofova, 1989). Although initially considered to function as a relay nucleus within ascending activating systems, increasing evidence on the neuronal heterogeneity of the PPN and its local synaptic organization, suggest that this high level of connectivity with functionally distinct neuronal systems underlies an integrative function rather than a role as a simple relay nucleus. An example of this heterogeneous connectivity is the way the PPN is integrated into basal ganglia circuits: distinct functional types of neurons in the PPN innervate basal ganglia and, in turn, basal ganglia projects back to PPN and innervate different neuronal populations. This remarkable interconnectivity has been the subject of previous reviews that have stressed that most structures of the basal ganglia project to, and receive inputs from, the PPN (Pahapill and Lozano, 2000; Mena-Segovia et al., 2004). In the present review we will discuss recent evidence on the heterogeneous distribution of neurochemical subtypes of neurons within the PPN and correlate this with data on their connectivity. We will make use of the large amount of information available that describe the anatomical relationship that the PPN maintains with the basal ganglia and that provide evidence of a topographical organization. We will also correlate such organization with the connectivity of the PPN with other neuronal systems to integrate a theory supporting functional domains in the PPN.

Neurochemical Diversity

It has now been widely agreed that the PPN is composed by a mixture of neurons of different sizes, of different neurochemical phenotype and with distinct connectivity. Cholinergic neurons represent a minority of the neurons in the PPN and are intermingled amongst a large number of GABAergic and glutamatergic neurons, which are heterogeneously distributed across its rostro-caudal axis (Mena-Segovia et al., 2009; Wang and Morales, 2009). A parasagittal view of the PPN illustrates the different distributions of the neuronal populations and therefore better represents the anatomical organization of the PPN. Using external landmarks such as the substantia nigra (SN) and the superior cerebellar peduncle, which maintain a constant spatial relationship with the PPN across different medio-lateral levels, it is possible to follow the distribution of the cell types that compose the PPN. GABAergic neurons are more densely concentrated in the rostral PPN, compared to cholinergic and glutamatergic neurons. In the rat they are detectable from the rostral border of the PPN (limiting the caudal part of the SN) and their density decreases dramatically at a level about 1.2–1.5 mm further caudal (Mena-Segovia et al., 2009; Figure 1). Such a drop in the density of GABAergic neurons coincides with a change in the cytoarchitecture and organization of cholinergic neurons: bipolar-shaped cholinergic neurons tend to be organized in a layer-like structure close to the SN, where GABAergic neurons are several times more abundant. Following the decline in the number of GABAergic neurons, rounded-shaped cholinergic neurons show a distinct configuration. Instead of lying in the layer-like arrangement, cholinergic neurons show an apparently random distribution and an increased number of processes. The change in density of cholinergic neurons, however, is not as marked as that of the GABAergic neurons or the glutamatergic neurons. In contrast to the rostral PPN, the caudal part of the nucleus has a larger proportion of glutamatergic neurons (Wang and Morales, 2009). Until now, no other neurochemical cell types have been identified although differences have been observed within each neuronal type in relation to the co-expression of other neurochemical markers and their firing properties.

FIGURE 1

Figure 1. Schematic representation of the distribution of distinct neuronal populations in the PPN. GABAergic neurons are highly concentrated in the rostral PPN, whereas cholinergic, glutamatergic (not shown), calbindin- and calretinin-expressing neurons are more abundant in the caudal PPN. The PPN was divided into 300 μm segments and cell density was evaluated throughout its rostro-caudal extent (Martinez-Gonzalez et al., 2009; Mena-Segovia et al., 2009). The difference in the rostro-caudal distribution of GABAergic neurons correlates with the differences in cytoarchitecture of the cholinergic neurons traditionally used to identify PPN regions (i.e., pars dissipata and pars compacta). As shown in this figure, the rostral PPN is an area of high neuronal density. SN, substantia nigra.

In different regions of the brain including hippocampus (Acsady et al., 1993; Somogyi and Klausberger, 2005), cortex (Staiger et al., 2004), and basal ganglia (Parent et al., 1996), neurons that express calcium-binding proteins have been shown to have distinct functional properties despite the fact that they may use the same transmitter. They have thus proved to be useful markers to distinguish sub-populations of neurons. Calcium-binding proteins have also been reported to be expressed by neurons in the PPN in monkeys (Cote and Parent, 1992; Fortin and Parent, 1999) and rats (Dun et al., 1995), and indeed, calbindin and calretinin are expressed by a similar number of neurons to that of cholinergic neurons in the rat PPN (Martinez-Gonzalez et al., 2009). Although they are rarely expressed in cholinergic neurons, they are commonly expressed by GABAergic and glutamatergic neurons suggesting functional subtypes of GABAergic and glutamatergic neurons.

Significant differences have been observed also in terms of the in vivo firing properties of PPN neurons. Cholinergic neurons show two types of firing patterns: slow-firing cholinergic neurons that are associated to the cortical upstate during slow oscillations, and fast-firing cholinergic neurons that are correlated to the cortical downstate during slow oscillations (Mena-Segovia et al., 2008). No neurochemical markers of these subtypes have been identified. Neurons that have been identified as non-cholinergic and have been assigned as putative glutamatergic neurons because they give rise to asymmetric synaptic contacts in their targets, also show two main subtypes: fast-firing neurons that are associated with the cortical slow oscillations (Mena-Segovia et al., 2008), and quiescent (or very slow firing) neurons whose firing is independent of the cortical activity (Ros et al., 2010). These putative glutamatergic neurons have a different axonal trajectory and pattern of innervation from those of cholinergic neurons, although some targets are shared by both types (notably, the basal ganglia). Other neurons that have not been characterized in terms of their neurochemical nature include tonic firing neurons and irregular firing neurons; it is likely that at least one of these subtypes are GABAergic (Ros et al., 2010). The correlation between neurochemical subtypes and electrophysiological properties recorded in in vitro experiments is more limited. Three types of neurons have been described on the basis of their membrane properties (A-current type, low-threshold spikes type and mixed A-current plus LTS type; Leonard and Llinas, 1994; Takakusaki et al., 1997; Saitoh et al., 2003), although this classification does not seem to be related to their neurochemical nature (and therefore not to their synaptic targets). The different membrane properties, however, are likely to underlie some of the functional differences within each cell subtype.

In summary, each main neuronal type in the PPN is composed of at least two subtypes; the PPN is thus a highly heterogeneous structure at the cellular, molecular, and electrophysiological levels. The different neuronal types are heterogeneously distributed in the PPN, perhaps delineating functional territories (rostral and caudal) determined by a greater density of GABAergic axons arising from the rostral PPN in contrast to a greater density of cholinergic and glutamatergic axons arising from the caudal PPN, thus producing contrasting effects on their target structures.

Internal Structure and Connectivity: The Case for Subdivisions of the PPN

The notion that the PPN is not homogeneous in terms of its cellular organization is not recent; the PPN was originally divided in pars dissipata (rostral) and pars compacta (caudal) on the basis of the density of cholinergic neurons (Olszewski and Baxter, 1982), which were believed to be the most representative, if not the only, neuronal type in the PPN (Rye et al., 1987). Other subdivisions included rostral, middle, and caudal thirds, and the inclusion of an area referred to as the midbrain extrapyramidal area (MEA) which receives a dense innervation from the basal ganglia but lies outside the cholinergic borders of the PPN (Steininger et al., 1992). While all of these subdivisions are based on identifiable characteristics in the anatomy of the PPN, it is perhaps the rostral and caudal division that is the most appropriate since it is based on the distribution of all known cell types rather than only the cholinergic neurons. Indeed, GABAergic neurons provide a unique distribution that correlates with the cholinergic pars dissipata and pars compacta subdivisions (Mena-Segovia et al., 2009). The rostro-caudal division is also the basis for many anatomical studies describing afferents and efferents to and from the PPN, as discussed in the following sections, but essentially does not rely on cell density (as the terms dissipata and compacta denote).

The idea of two functionally distinct regions of the PPN is supported by the distribution of its cell types defined on the basis of neurochemistry and the connectivity of the PPN with other neuronal systems. Thus, two neurochemically distinct projections arising from rostral and caudal PPN diverge, innervating distinct structures, but also converge in others. This functional dichotomy seems to be locally regulated, as suggested by the evidence of axonal collaterals of PPN neurons. Thus, a local synaptic network has been identified after tracing the axons of individually labeled neurons (Mena-Segovia et al., 2008; Ros et al., 2010). Both cholinergic and non-cholinergic neurons contribute to this connectivity, although the number of axonal varicosities was found to be greater in cholinergic neurons. Interestingly, a large proportion of PPN projection neurons have axons that travel within the PPN in a rostro-caudal direction, providing local innervation that synaptically links the rostral and the caudal portions. This evidence of a local network of heterogeneous neurochemical nature supports the role of the PPN as an integrator between its input and output systems.

Efferent Connectivity

Neurons of the PPN give rise to long axons that innervate several targets. The longest axons arise from cholinergic neurons and give rise to as many as five or six collaterals that innervate the basal ganglia, thalamus, tectum, and lower brainstem, among other regions (Mena-Segovia et al., 2008). The axons of non-cholinergic neurons are more restricted in terms of length and number of collaterals (typically two; Ros et al., 2010). Single-cell labeling experiments, have demonstrated that both cholinergic and non-cholinergic neurons project preferentially to the basal ganglia and that several divisions of the basal ganglia are innervated. Tracer studies have also produced extensive data on the connections of neurons in the PPN (Table 1).

The entopeduncular nucleus (EP, in rat and cat, equivalent to the internal segment of the globus pallidus or GPi in the monkey) receives input from the PPN (Saper and Loewy, 1982; Jackson and Crossman, 1983; Woolf and Butcher, 1986; Shink et al., 1997), and these projections have an excitatory influence on EP neurons in the rat and cat (Gonya-Magee and Anderson, 1983; Scarnati et al., 1988). A species-specific difference seems to exist regarding the density of these projections, since they have been reported to be larger in the monkey than in the cat (Edley and Graybiel, 1983). In the monkey, tracer injections in the GPi give rise to a large number of retrogradely labeled PPN neurons in the rostral PPN, around 40% of which are cholinergic. In contrast, a restricted GPe injection produced a smaller number of labeled neurons in the PPN (Charara and Parent, 1994). This difference was confirmed by anterograde labeling, Phaseolus vulgaris leucoagglutinin injections in the PPN give rise to a much higher density of anterogradely labeled fibers in the GPi than in the GPe (Lavoie and Parent, 1994).

Other Ascending Projections

Individual cholinergic neurons of the PPN project to the superior and inferior colliculi in the rat (Mena-Segovia et al., 2008). The large majority of the cholinergic inputs to the inferior colliculus arise from the ipsilateral PPN and to a less extent, from the LTD. These projections arise from cholinergic neurons that are located in the caudal PPN and include a subpopulation that project to both the ipsilateral and contralateral inferior colliculus in the guinea pig (Motts and Schofield, 2009). The superior colliculus receives cholinergic and non-cholinergic innervation from the PPN in the rat and cat (Beninato and Spencer, 1986; Hall et al., 1989). These afferents arise mainly from the caudal PPN (Beninato and Spencer, 1986). PPN neurons projecting to the superior colliculus, as identified by antidromic stimulation, are segregated into two groups: those that are sensitive and those that are insensitive to physiological sensory stimuli (Krauthamer et al., 1995). A small group of neurons located in the rostral PPN have collaterals that innervate the superior colliculus and the LM-Sg in the cat (Hoshino et al., 2004). In the rat, other ascending targets include the mesopontine rostromedial tegmental nucleus (RMTg; Jhou et al., 2009).

In the forebrain, the magnocellular preoptic area (MgPA) and the nucleus basalis magnocellularis receive afferents from the PPN (Semba et al., 1988; Losier and Semba, 1993). The posterior lateral hypothalamus receives cholinergic input from PPN neurons that are scattered throughout the rostro-caudal axis, and GABAergic innervation from neurons that are concentrated in the rostral PPN; the neurochemical nature of the majority of the projection neurons was not identified (Ford et al., 1995).

In summary, the ascending projections from the rostral PPN preferentially innervate the EP/GPi, SN and the lateral hypothalamus in the rat, cat, and monkey. In contrast, ascending projections from the caudal PPN innervate the thalamus, STN, VTA, SC, and IC.

Afferent Connectivity

Although the information available on the afferent innervation to the PPN is not as abundant and detailed as it is with regards to its efferents, it is clear that the PPN receives a heterogeneous modulation arising from functionally diverse areas of the brain. Thus, neurons in the PPN receive afferents from structures that include the cortex, thalamus, hypothalamus, pons, cerebellum, medulla, spinal cord, and the basal ganglia (Saper and Loewy, 1982; Semba and Fibiger, 1992; Table 2).

TABLE 2

Table 2. Afferent connectivity of the pedunculopontine nucleus.

The PPN receives a direct input from the cerebral cortex arising from distinct frontal lobe areas involved in motor control in the monkey. These convergent inputs seem to target the dorsal and caudal PPN areas (Matsumura et al., 2000). In the rat, these afferents have also been demonstrated, although they seem to be less abundant (Semba and Fibiger, 1992). They arise also from the medial prefrontal cortex (Sesack et al., 1989). In addition, cholinergic PPN neurons receive afferents from the primary auditory cortex in guinea pigs (Schofield and Motts, 2009).

Tracer studies show that the EP sends projections that innervate PPN neurons in the rat (Semba and Fibiger, 1992) and in the monkey (Shink et al., 1997). In the latter, the GPi afferents preferentially target NADPH diaphorase-negative neurons in the rostral PPN, establishing symmetric synapses with proximal dendrites. GP afferents to the PPN arise from the caudal GP, in contrast to the rostral GP that projects to the STN (Moriizumi and Hattori, 1992). GPi axons that innervate PPN neurons arise from type I neurons that are abundant in the center of the GPi (Parent et al., 2001), although some authors differ on this, finding retrogradely labeled neurons in the whole GP after retrograde tracer injection in the rat STN (Smith et al., 1990). This is particularly relevant because the PPN is involved into a circuit that involves the STN and GP, one of the principal outflows of the basal ganglia.

In summary, the PPN receives afferents from the basal ganglia, cortex, thalamus, cerebellum, forebrain, spinal cord, pons, and the contralateral PPN. The rostral PPN receives inhibitory input from the SN and the EP/GPi. The caudal PPN receives inputs from the dorsal raphé and the motor cortex.

Functional Implications of Topography

The data arising from the connectivity studies show that a significant number of structures have a selective relationship with distinct regions within the PPN. This is clearly evident from the analysis of retrograde and anterograde tracing studies showing the distribution of PPN projecting neurons, although less clear with regards to the distribution of the PPN afferents. Nevertheless, in the case of PPN inputs, two important neuronal systems, the basal ganglia and the cortex, seem to contact neurons in distinct regions of the PPN. Thus, the GABAergic output neurons of the basal ganglia, arising in the SNr and EP/GPi, mainly contact neurons located in the rostral PPN. In contrast, neurons in the cortex and the dorsal raphé preferentially innervate neurons in the caudal PPN. In terms of its efferents, the rostral PPN projects to the SNr, SNc, GPi, and the hypothalamus. In contrast, the caudal PPN projects to the STN, the VTA, the thalamus, and the superior and inferior colliculi (Figure 2).

FIGURE 2

Figure 2. Summary of the topographical distribution of the connectivity in the PPN. The rostral PPN, which is predominantly GABAergic, maintains interconnections with the GABAergic output of the basal ganglia. In contrast, the caudal PPN, where cholinergic and glutamatergic neurons are more abundant, receives input from the cortex and dorsal raphé and projects to the thalamocortical systems, STN and locomotor regions. Only major inputs and outputs, and those structures whose connectivity with the PPN is topographically organized, are depicted in this scheme. EP, entopeduncular nucleus; GPi, internal segment of the globus pallidus; IC, inferior colliculus; SC, superior colliculus; SN, substantia nigra; STN, subthalamic nucleus; VTA, ventral tegmental area.

The differences in connectivity suggest that there is a functional reciprocity in different areas of the PPN with regards to its inputs and outputs. These differences reveal that, (1) the rostral PPN, which contains a significantly larger number of GABAergic neurons, is interconnected with the structures that provide the GABAergic output from the basal ganglia, therefore suggesting a close functional relationship with basal ganglia operations; and (2) the caudal PPN, which contains a larger number of cholinergic and glutamatergic neurons, receives information from cortex and dorsal raphé, and projects to targets in the thalamus and colliculi, suggesting a close relationship with the modulation of brain states mediated through thalamocortical systems. Moreover, it is also the caudal PPN that projects to the STN and to the brainstem locomotor regions involved in gait and posture and the modulation of the muscular tone across different brain states.

Conclusion

The PPN is subdivided in two functionally distinct regions: the rostral portion, which is predominantly inhibitory and interconnected with the basal ganglia, and the caudal portion, which is predominantly excitatory and closely related to arousal and motor systems. These two functionally distinct areas are locally regulated and synaptically linked by the local axon collaterals of cholinergic and non-cholinergic neurons. The extent of the interaction between these two regions remains to be determined, but will help to elucidate the common mechanism by which PPN neurons seem to participate in a wide range of behavioral functions.

Conflict of Interest Statement

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

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